Cancer causes 1 of every 4 deaths in the US, and it is a critical research goal to develop fundamentally new, clinically useful anticancer drugs. The enediynes are the most potent, highly active anticancer agents in existence today, and their utilities as anticancer drugs have been demonstrated clinically. A great challenge is to develop ways to prepare enediynes and their structural analogs for further mechanistic studies and clinical developments. We propose (1) to continue to study the chemistry, biochemistry, and genetics of the production of C-1027 and neocarzinostatin (NCS) as models for enediyne biosynthesis and (2) to apply combinatorial biosynthesis methods to the enediyne biosynthetic machinery for the production of novel anticancer drugs. Our hypotheses are: (1) the availability of the C-1027 and NCS as well as the maduropeptin (MAD) biosynthetic gene clusters will provide a unique opportunity to investigate the molecular basis of enediyne biosynthesis by a comparative genomics approach;(2) characterization of the novel enzymes involved in C-1027 and NCS biosynthesis will make fundamental contributions to mechanistic enzymology and natural product chemistry;(3) systematic application of combinatorial biosynthetic methods to the C-1027 and NCS biosynthetic machinery will result in the production of a novel enediyne library;and (4) engineering of C-1027, NCS, and their analogs as cancer-targeting peptide (CTP)-containing chromoproteins will provide a new strategy to deliver the enediynes to specific cancer cells. The long-term goals for this project are to understand at the molecular level how microorganisms biosynthesize complex natural products such as the enediynes and to apply combinatorial biosynthesis methods to the enediyne biosynthetic machinery for anticancer drug discovery. The specific aims for this proposal are: (1) functional analysis of the genes governing the biosynthesis of the enediyne core;(2) functional analysis of the genes governing the biosynthesis of the periphery moieties of the enediynes;(3) engineering of novel enediynes by applying combinatorial biosynthesis methods to the enediyne biosynthetic machinery;and (4) engineering of C-1027, NCS, and their analogs as CTP-containing chromoproteins. The outcomes of these studies will unveil new chemistry and biology for natural product biosynthesis, enrich the toolbox for combinatorial biosynthesis, and potentially lead to the discovery of new anticancer drugs.